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Iseaed  April  20, 1911. 


U.  S.  DEPARTMENT  OF  AGRICULTURE, 

BUREAU  OF  ANIMAL  INDUSTRY.— BUL^ETIN  135. 

A.  D.  MELVIN,  Chief  op  Bureau. 


i  COMPARATIVE  STUDY  OF  METHODS  OF 
EXAMINING  FECES  FOR  EVIDENCES 
OF  PARASITISM. 


V 


BY 


MAURICE  C.  HALL, 
Assistant  Zoologist,  Zoological  Division. 


4t  iiOi  AHOBUII 

JAN  4    1943 
L,l  ^  RAR  Y 


WASHINGTON  : 

GOVERNMENT   PRINTING  OFFICE. 
1911. 


LsMueil  April  JU,  I'Jll. 

U.  S.  DEPARTMHNT  OF  AGRICULTURE, 

BUREAU  OF  ANIMAL  INDUSTRY —Bui.i.ktin  135. 

A.   D.  MELVIN,  C.HifcK  ok  Bureau. 


A  COMPARATIVE  STUDY  OF  METHODS  OF 

EXAMINING  FECES  FOR  EVIDENCES 

OF  PARASITISM. 


BY 


MAURICE  C.  HALL. 

Afisi.stant  Zooloffist,  Zoological  Dirisivn. 


WASHINGTON  : 

GOVHRNMHNT    I'RINTING    OFFICE. 
1^1L 


THE  BUREAU  OF  ANIMAL  INDUSTRY. 


Chief:  A.  D.  Melvin. 
Assistant  Chief:  A.  M.  Farbington. 
Chief  Clerk:  Charles  C.  Carroll. 

Animal  Husbandry  Division:  George  M.  Rommel,  chief. 
Biochcmic  Division:  M.  Dorset,  chief. 
Dairy  Division:  B.  H.  Rawl,  chief. 

Inspection  Division:  Rice  P.  Steddom,  chief;  Morris  Wooden,  R.  A.  Ramsay, 
and  Albert  E.  Behnke,  associate  chiefs. 

Pathological  Division:  John  R.  Mohler,  cliief. 
Quarantine  Division:  Richard  W.  Hickman,  chief. 
Zoological  Division:  B.  H.  Ransom,  chief. 
Experiment  Station:  E.  C.  Schroeder,  superintendent. 
Editor:  James  M.  Pickens. 

zoological  division. 

Chief:  B.  H.  Ransom. 

Assistant  Zoologists:  Albert   Hassall,   Harry  W.   Graybill,   and   Maurice 
C.  Hall. 
Junior  Zoologists :  Howard  Crawley  and  Winthrop  D.  Foster. 

2 


LETTER  OF  TRANSMITTAL. 


UXITKD  StATKS  DhPAIMMKN  1    Ol'    AcHfK  ir/nKK. 

lit  HKAi  OK  Animal  Ixdistkv, 
Washington,  I).  C,  Jannanj  17,  U)l  1. 
Sir:  I  have  tlie  honor  to  tniiisinit  the  acc-onipanying  iiianiiscript 
of  an  article  entitled  "A  Coin])arative  Study  of  Methods  of  Examin- 
in<j:  Feces  for  Evidences  of  Parasitism."  by  Mr.  Maurice  C.  Hall. 
Assistant  Zoologist  in  the  Zoological  Division  of  this  bureau,  with  the 
reconunendation  that  it  be  published  as  a  bulletin  of  this  bureau. 

!Mr.  Hall  has  devised  a  method  of  examining  feces  b}'  means  of 
which  the  presence  or  absence  of  parasites  infesting  the  alimentary 
tract  may  be  rapidly  and  accurately  determined.  Various  methods 
of  fecal  examination  are  in  vogue  among  medical  and  zoological 
investigators,  and  these  are  all  critically  compared  with  Mr.  Hall's 
method,  which  is  shown  to  yield  superior  results. 
Respectfully, 

A.  1).  Mklvix, 
Chief  of  Bureau. 
Hon.  James  Wilson, 

Secreturij  of  A</)i<uUure. 


Digitized  by  tine  Internet  Archive 

in  2007  with  funding  from 

IVIicrosoft  Corporation 


http://www.archive.org/details/comparativestudyOOhalliala 


CONTENTS. 


Page 

Iiilrodiu'tion 7 

Methods  of  initToscopic  examination 7 

Smear  method 7 

Sedimentation  method N 

Burette  method '*^ 

('entril'ufi;e  methods. ^ 

Sieve  methods ^ 

(iasteiger's  filter  method !• 

Bass's  salt  solution  method 9 

iiass's  calcium  cidorid  centrifujje  method 10 

Garrison's  calcium  chlorid  sedimentation  metiiod 10 

Wellman's  sodium  acetate  centrifuge  method II 

Pepper's  adhesion  method 11 

Telemann's  ether  hydrochloric  acid  method II 

Culture  methods 12 

Purpose  of  methods 12 

Advantages  of  concentration I-* 

Pniperties  used  in  concentration 1:5 

Physical  properties I:i 

Chemical  properties II 

Biological  properties 11 

The  theoretical  ideal I ;"> 

Practical  objections 15 

Gross  examination 1  •'> 

The  writer's  method Hi 

Comminution  of  feces 10 

Sieving i  S 

Sedimenting  and  centrifuging 21 

Preparation  oi  slides 21 

('oncentralion  obtained  by  the  use  of  sieves 22 

Summary  of  method 22 

Collecticm  index 2:1 

Tests  of  un])ublished  methods 2^ 

Com  para  ti\e  tests  of  the  writer's  method  and  other  methods 2') 

Smear  method 2') 

Sedimentation  method 25 

Burette  method 25 

Gasteiger's  filter  method 2(1 

Bass's  calcium  chlorid  centrifuge  nn^thod 20 

Garrison's  calcium  chlorid  sedimentation  method 2S 

Telemann's  ether  hydrochloric  acid  method 2S 

Culture  methods .!() 

Objections  to  certain  methods ;;  1 

Injury  to  parasites ;51 

Limited  application  or  incommeiisurale  rcsidts ;{2 

Disinfection  of  feces :i2 

Summary ;);5 

Adaptability  of  method :VA 

Economy  of  method :M 

Conclusion :i  1 

Bibliography ,{5 


ILLUSTRATION. 


Page 
Fig.  1.  Apparatus  used  in  the  writer's  method  of  examining  feces 17 

G 


A  COMPARATIVE  STUDY  OF  METHODS  OF  EXAMINING 
FECES  FOR  EVIDENCES  OF  PARASITISM. 


INTRODUCTION. 

Havin*;  occasion  to  make  numerous  examinations  of  feces  for  evi- 
dences of  parasitic  infection,  the  writer  lias  clevoted  some  time  to 
developing^  a  satisfactoiy  method  which  would  be  capi)l)le  of  <reneral 
application  in  routine  examinations  and  which  Avould  give  the  surest 
results  in  the  shortest  time,  one  which  would  detect  any  existing  in- 
fection, even  though  light,  in  any  kind  of  feces,  so  far  as  the  existing 
limitations  of  fecal  examinations  permit.  The  ditferent  methods 
which  have  been  used  and  advocated,  most  of  them  being  methods 
used  in  examining  human  feces,  were  tested  on  feces  of  various  kinds. 
This  paper  deals  with  the  results  of  these  tests.  The  method  which 
gave  the  most  satisfactory  results  and  which  is  hereafter  refeircd 
to  as  the  writer's  method  is  a  modification  of  previous  metliods. 

Fecal  examinations  are  of  two  sorts — first,  examination  with  tlie 
naked  eye  for  gross  evidence  of  infection  in  the  sha]ie  of  entire  para- 
sites or  fragments,  and,  second,  microscopic  examination  for  paia- 
site  eggs,  embryos,  etc.  The  first  is  vei-y  commoidy.  jierhaps  wrongly. 
neglected;  the  second  is  commonly  used  and  will  be  considei'cd  lii-st 
here. 

METHODS    OF    MICROSCOPIC    EXAMINATION. 
SMEAK    MKTIKM). 

The  microscopic  method  which  is  commonly  u.s<m1  and  advocated 
is  what  may  be  called  the  smear  method.  A  small  particle  of  feces 
is  taken  up  with  a  toothpick  or  stirring  rod.  smeared  on  a  glass  slide 
in  a  drop  of  water  or  salt  solution,  covered  with  a  cover  glass,  and 
examined  under  the  miscroscope.  This  method  is  the  oldest  and  the 
simplest,  can  be  used  under  almost  any  conditions,  and.  in  that  it  calls 
for  no  equipment  beyond  a  toothpick,  a  slide  and  cover  glass,  and  a 
microscope,  is  also  the  most  inexpensive.  It  is  usually  coiisidcrcil 
satisfactory,  providing  a  numln'r  of  slide  preparations,  commonly 
10,  are  examined.     This  method  has  been  repeatedly  commended  by 


8  METHODS   OF   EXAMINING  FECES   FOR  PARASITISM. 

Stiles  (1902hh,  19031,  lOOfia),  is  one  commonly  used  by  physicians 
(Jones,  1907,  et  al,),  and  was  used  by  Garrison,  Ransom,  and  Steven- 
son (1903a),  and  Stiles  and  Garrison  (1906a)  in  Washington,  and  by 
Garrison  (1908)  in  Manila. 

SEDIMENTATION    METHOD. 

The  simplest  modification  of  this  method  consists  in  allowino;  the 
feces,  if  sufficiently  fluid,  to  settle,  and  then  examining  the  sediment ; 
if  the  feces  are  too  solid  for  this,  large  or  small  amounts  are  washed 
in  a  sufficient  quantity  of  water,  decanted  as  long  as  any  matter  will 
float,  and  the  sediment  finally  examined.  Stiles,  in  the  papers  just 
cited,  Braun  (Braun  and  Liihe,  1909),  Garrison  (1910),  Letulle 
(1905),  Jones  (1907),  and  others  have  advocated  this  sedimentation 
method. 

BURETTE  METHOD. 

T  am  informed  by  Dr.  Ransom  that  one  writer,  in  an  article  to 
which  I  have  not  a  reference  at  present,  has  varied  the  ]5receding 
method  by  taking  the  sediment  from  the  bottom,  through  a  stopcock, 
a  method  designated  in  this  paper  as  the  burette  method. 

CENTRIFUGE   METHODS. 

Another  variation  of  the  sedimentation  method  is  to  use  a  centri- 
fuge for  the  purpose  of  giving  a  more  rapid  and  certain  concentra- 
tion of  material.  Pepper  (1908)  states  that  he  has  found  repeated 
centrifuging,  in  which  the  sediment  is  shaken  up  each  time  with 
some  fresh  water,  very  useful.  This  is  the  process  commonly  known 
as  washing.  Bass  (1909)  uses  the  same  device  as  part  of  his  method. 
Braun  (Braun  and  Liihe,  1909)  notes  that  under  certain  circum- 
stances the  sediment  may  be  centrifuged  after  sedimenting  and 
decanting.  He  does  not  state  under  what  circumstances  this  is  to 
be  done.  Letulle  (1905)  admits  the  use  of  the  centrifuge  if  needed, 
but  objects  to  it  on  the  ground  that  certain  eggs  suffer  mechanical 
injury  from  its  use.  Stiles  (190"2hh,  19031)  states:  "The  centrifuge 
does  not  appear  to  be  of  any  special  value  in  fecal  examinations." 

SIEVE  INIETIIODS. 

Another  variation  of  technique  consists  in  the  use  of  one  or  more 
sieves  or  screens  to  take  out  coarse  particles  of  undigested  food  and 
similar  objects.  Stiles  (1902hh,  19031)  gives  a  qualified  indorse- 
ment of  this  method  in  the  following  terms : 

In  case  an  muisually  large  amount  of  larjje,  coarse  niaterinl  is  present  in  the 
feces,  it  is  sometimes  convenient  to  pour  the  entire  mass  tlirousli  a  sieve,  re- 


GASTEIGER  S    AND    BASS  S    METHODS.  9 

jectiiiK  the  portion  left  in  the  sit'vc;  or  to  w.isii  tin'  fwt's  in  a  siovc.  liojdin;; 
tlie  latter  nndor  water.  As  a  rule,  liowever.  Hie  sieve  is  not  very  useful  in  fecal 
examinations. 

On  the  other  hand,  the  use  of  the  sieve  receives  tiiiqualified  iudoise- 
nient  from  Cobb  (1904).  In  exan»iniiig  sheep  feces  for  fluke  e<;<ijs 
lie  boils  a  pellet  or  part  of  a  pellet  of  sheep  dung  iu  water  for  a 
few  minutes;  puts  it  in  a  hemispherical  brass  sieve  of  80  to  100 
meshes  to  the  inch,  the  sieve  being  in  a  watch  glass  full  of  water,  and 
itself  standing  partly  full  of  water;  brushes  the  pellet  through  tiie 
sieve  by  means  of  a  sable  brush;  sediments  the  fluid  containing  the 
fecal  matter  which  passes  through  the  sieve,  and  repeatedly  removes 
the  supernatant  muddy  fluid  with  a  pipette  until  water  poured  on 
the  sediment  remains  clear:  and  then  brushes  this  sediment  through 
a  sieve  of  miller's  silk  bolting  cloth  of  five  meshes  to  the  millimeter. 
The  water  which  passes  through  the  silk  sieve  is  then  examined  for 
fluke  eggs. 

Bass  (190!>),  Teleiuaun  (lOOS),  and  Garrison  (1910)  use  a  sieve 
of  some  sort  in  nuiking  examinations  of  feces. 

gastei(;kr's  filtek  method. 

Gasteiger's  (1004)  method  is  an  inversion  of  the  methods  where 
a  centrifuge  and  sieve  are  used.  AMiereas  with  the  latter  the  object 
is  to  screen  out  foreign  particles  larger  than  parasites  and  eggs  and 
seek  for  the  parasitic  material  at  the  bottom  of  the  centrifuge  tube. 
Gasteiger,  in  his  search  for  eggs  of  Ascat'is  in  the  stalls  of  cattle, 
soaks  the  nuHun-e.  straw,  and  mud  in  water;  filters  this  water  through 
some  unspecified  sort  of  filter,  and  examines  the  residue,  not  the  fil- 
trate, for  eggs.  This  accomplishes  to  a  lesser  degree  the  same  thing 
that  is  accomplished  by  sedimentation,  sieving,  and  centriftiging — 
it  gives  a  concentration  of  material. 

HASs's   SAI/r   SOMTION    METHOD. 

Bass  (1006)  proposed  a  method  which  consisted  in  putting  a  quan- 
tity of  feces  in  a  \ial  three-fourths  fidl  of  a  nine-tenths  saturated  so- 
lution of  soflium  chlorid,  shaking  this  well,  allowing  it  to  settle,  and 
then  examining  a  drop  from  the  surface  of  the  fiuid.  This  method 
was  proposed  for  the  examination  of  liuuian  feces  for  hookwoiMu  eggs. 
According  to  Bass,  all  the  eggs  rise  to  the  surface  and  large  numbers 
may  be  found  in  one  drop.  Of  course  this  method  depends  on  the 
specific  gravity  of  the  parasite  egg  being  less  than  that  of  the  solu- 
tion employed.  Waddell  (1010)  states  that  if  an  examination  of 
three  slides,  presumably  snu'ar  j)rei)arations.  did  not  disclose  human 
hookworm  eggs,  this  method  of  Bass's  was  used,  and  says:  "  By  this 
method  from  30  to  40  minutes  could  be  saved  per  stool  examined.*" 
7S939°— Bull.  IG.'j— 11 2 


10  METHODS    OF    EXAMINING   FECES    FOR   PARASITISM. 

KASS'S   CALCIUM    CHLORID   CEXTKIFUGE    ^METHOD. 

Afterwards  Bass  (1909)  modified  the  above  method  as  follows: 
A  quantity  of  feces  is  diluted  with  water,  1  in  10,  and  strained 
throup;h  gauze  to  get  rid  of  coarse  particles.  What  comes  through 
is  centrifuged,  the  fluid  poured  off,  the  centrifuge  tube  refilled  and 
the  fresh  material  and  the  old  sediment  centrifuged  again,  thereby 
constantly  adding  to  the  total  sediment,  until  all  the  diluted  feces 
have  been  used.  The  sediment  is  rew ashed  several  times  until  all 
matter  that  can  be  w^ashed  out  in  this  manner  is  removed.  Then  a 
calcium  chlorid  solution  of  a  specific  gravity  of  1.050  is  substituted  for 
the  water.  This  disposes  of  everything  having  a  specific  gravity 
below  1.050,  and  the  sediment  may  be  examined  at  this  point.  If 
much  sediment  remains,  the  heavier  matter  may  be  removed  by  cen- 
trifuging  with  a  calcium  chlorid  solution  having  a  specific  gravity  of 
1.250.  In  this  solution  the  eggs  come  to  the  top  and  a  few  drops  from 
the  surface  may  be  removed  and  examined,  or,  better,  some  of  the  top 
fluid  may  be  poured  off,  diluted  with  water  sufficiently  to  bring  the 
specific  gravity  below  1.050,  and  centrifuged.  The  sediment  Avill 
now  contain  most  of  the  eggs  that  Avere  in  the  original  amount  of 
feces  and  may  all  be  put  on  a  slide  and  examined. 

garrison's    CALCIUM    CHLORID    SEDUAIENTATION    METHOD. 

Garrison  (1910)  has  outlined  a  method  which  is  essentially  a  modi- 
fication of  Bass's  (1909)  method.     According  to  GaiTison — 

The  specific  gravity  of  the  ova  is  from  1.050  to  1.100  (old  eggs  sometimes 
higher,  according  to  Bass).  If  the  stool  be  liberally  diluted  with  tap  water 
the  mixture  has  a  considerably  lower  specific  gravity,  which  varies,  of  course, 
with  the  character  of  the  stool,  but  is  usually  about  1.005,  so  that  the  ova,  to- 
gether with  the  heavier  sediment,  sink  to  the  bottom.  [Allow  to  stand  for  an 
hour  or  more;  decant  and  add  fresh  water  repeatedly  until  soluble  matter  is 
washed  out.]  At  any  time  during  the  sedimentation,  but  preferably  after  the 
specimen  has  been  washed  a  few  times,  the  coarser  material  may  be  removed 
by  straining  and  washing  the  sediment  through  a  fine  wire  gauze,  using  a 
small,  strong  jet  of  water.  To  completely  wash  a  specimen  until  the  super- 
natant water  is  clear  may  require  quite  a  number  of  sedimentations,  and  it 
may  be  desirable  to  continue  the  process  throughout  one  or  more  days.  *  *  * 
Sometimes  the  feces  contain  heavy,  gritty,  solid  material,  which  is  particularly 
annoying  in  nuiking  slide  preparations.  The  specific  gravity  of  much  of  this 
material  is  sufficiently  higher  than  that  of  the  eggs  to  allow  the  use  of  a  solu- 
tion with  a  siiecific  gravity  between  the  two  which  will  float  the  eggs  to  the 
surface  and  allow  the  heavy  sediment  to  sink.  This  may  be  done  by  suspend- 
ing the  specimen,  preferably  after  it  has  been  well  washed,  in  a  solution  of  cal- 
cium chlorid  containing  350  grams  to  the  liter  of  water,  which  gives  a  specific 
gravity  of  about  1.200.  (A  saturated  solution  of  the  commercial  salt  has  about 
the  same  specific  gravity.)  The  top  layer,  containing  eggs  and.  the  lighter 
debris,  is  decanted,  leaving  the  heavy  sediment  behind. 


WELLMAN  S,   PEPPER  S,   AND   TELKMANN   S    METHODS.  11 

WELLMAN's  SODIIM    ACETATK  (  KNTHII  r<;K    METHOD. 

Wellniaii  (1910)  oniploys  a  method  wliicli  apin'sirs  to  lu'  a  modifica- 
tion of  Bass's  (11)09)  method,  but  as  it  is  not  clear  just  what  Welinian 
means  by  his  statement  of  his  method,  the  writer  has  not  tested  it. 
His  method,  which  is  for  hookworm  e<;gs,  is  <>;iven  as  foHows: 

The  princii)Ie  involved  was  first  oinployed  iiidoiKMuUMitly  l».v  tlio  writer  niid  l)y 
Dr.  Hass.  of  New  Orleans.  Dr.  Hass  beinjr  the  tirst  to  itiihlish  his  results.  Our 
own  method  is  to  use  two  solutions  of  sodium  acetate  of  sinvilic  ;rravities  of 
1.050  and  1.250.  resi>e<'tively.  A  i>ortion  of  snspectetl  feces  is  mi.xed  with  one  of 
these  solutions  and  centrifuj;ed  for  about  10  seconds,  the  li(iuid  decanted,  then 
the  other  solution  is  poured  on  and  nnxetl  gently,  the  whole  apiin  centrifujied, 
and  this  process  rei)eated  until  tlie  (>frKS  are  all  in  one  layer,  the  sand  and  other 
heavy  ingredients  of  the  feces  remaining  below  and  the  light  tiocculent  com- 
ponents lying  above  the  zone  in  which  the  eggs  remain.  With  this  technique 
one  slide  contains  as  many  ova  as  50  or  100  slides  l)y  the  ordinary  method.  Dr. 
Bass  uses  calcium  chlorid  for  his  solutions,  but  the  writer  lias  not  been  abl(> 
to  employ  this  salt  satisfactorily  on  account  of  its  hygroscopic  properties. 

It  is  unfortunate  that  Welhnan  does  not  state  just  why  the  liyji^ro- 
scopic  properties  of  calcium  chlorid  are  objectionable,  as  Bass  em- 
ploys this  salt  on  accoimt  of  these  properties. 

pepper's  adhesion  method. 

An  interesting  application  of  technique  to  a  particular  case  is  that 
of  Pepper  (1908).  He  takes  advantage  of  the  stickiness  which  he 
finds  to  be  a  property  of  human  hookworm  eggs  in  the  following 
manner:  Washed  and  sedimented  feces  are  put  on  a  slide  for  a  few 
minutes  and  then  innnersed  gently  in  water;  on  lifting  it  out  the 
eggs  are  foinid  adhering  after  everything  else  has  been  washed  away. 
This  process  may  be  repeated  and  numerous  eggs  collected.  Pepper 
does  not  find  the  same  stickiness  in  the  case  of  Ascarls,  Tvichuris^  or 
T(pnia  eggs. 

Stiles  (1910)  has  given  the  smear  method  and  the  methods  of  Pep- 
per (1908)  and  Bass  (1909)  for  the  examination  of  human  feces  for 
evidence  of  hookworm  infection. 

TELEMANX's    ETHER    HYDROCHLORIC    ACID    METHOD. 

Another  modification  of  method  consists  in  using  chemical  as  well 
as  physical  means  to  secure  a  concentration  of  j)arasitic  material. 
Telemann  (1908).  who  appears  to  have  been  the  first  to  use  chemical 
methods  in  examining  feces  for  j)arasite  ^^,1*1.^,  uses  the  following 
technique:  Small  particles  are  taken  from  a  niunber  of  places  in  the 
feces  tmder  investigation  and  shaken  u|)  in  a  mixtiu'c  of  c(|ual  jiarts 
of  ether  and  ptuv  hydrochloric  acid  in  a  reagent  glass.  (  1  believe 
that  I  have  had  slightly  better  results  from  adding  ether  iirst.  stirring 
well,  and  then  adding  the  hydrochloric  acid.)     The  ether  dissolves 


12  METHODS   OF   EXAMINING   FECES   FOE   PARASITISM. 

the  neutral  fats  and  free  fatty  acids,  while  the  hydrochloric  acid  dis- 
solves the  albuminous  matter,  as  casein,  etc.,  soaps,  mucin,  phosphates, 
and  various  calcium  salts.  This  mixture  is  then  filtered  through  a 
fine  hair  sieve  to  eliminate  large  particles,  and  the  liquid  is  then  cen- 
trifuged  about  a  minute.  In  the  centrifuge  tube  will  be  found  three 
layers,  an  upper  one  of  fats  dissolved  in  ether,  a  middle  one  of  bac- 
teria and  snuill  detritus  in  the  acid,  and  finally  a  sediment  of  small 
food  particles,  mostly  cellulose  and  muscle  fibers,  and  parasite  eggs. 
The  parasite  eggs,  being  relatively  more  concentrated  in  the  sediment 
than  in  the  original  feces,  can  readily  be  found  in  the  usual  manner 
by  microscopic  examination  of  a  slide.  This  method  has  been  tested 
and  commended  by  Quadflieg  (1909),  who  notes,  however,  that  it 
should  be  supplemented  by  the  smear  method,  as  the  latter  seems  to 
be  superior  in  the  case  of  certain  parasites.  Pfister  (1909)  commends 
the  method  as  being  satisfactory  in  searching  for  Bilharzia  eggs. 

CULTURE    METHODS. 

A  final  modification  of  technique  is  to  use  culture  methods,  the 
feces  being  kept  moist  and  warm,  and  after  a  proper  interval  being 
examined  for  embryos  which  have  developed  and  escaped  from  the 
eggs.  Under  certain  conditions  this  method  gives  a  concentration  of 
material  on  the  glass  receptacle  or  in  water  contained  in  depressions 
on  the  surface  of  the  feces. 

PURPOSE   OF   METHODS. 

Inasmuch  as  the  writer's  method  is  a  variation  of  some  existing 
methods  already  outlined,  it  may  be  in  order  at  this  point  to  find  a 
basis  for  the  selection  of  a  method  by  considering  what  the  purpose  of 
the  microscopic  examination  and  the  reason  for  any  method  may  be. 

Obviously,  the  purpose  of  the  examination  is  to  detect  parasites  and 
their  eggs  in  the  feces  if  they  are  present.  The  evident  w^ay  to  ac- 
complish this  is  to  examine  the  feces  on  a  microscopic  slide  prepara- 
tion after  they  are  so  thinned  as  to  permit  of  exauiination.  Every 
method,  no  matter  how  complicated,  includes  these  essential  steps. 
The  smear  method  accomplishes  this  in  the  minimum  numljer  of 
steps:  fluid  feces  are  examined  at  first  hand;  solid  feces  are  rendered 
fluid  and  then  examined  or  are  mounted  in  a  drop  of  water. 

ADVANTAGES  OF  CONCENTRATION. 

The  purpose  of  all  modifications  of  this  simple  proceeding,  as  the 
preceding  sketch  shows,  is  to  attain  one  object— the  concentration  of 
parasites  and  their  eggs  in  order  to  facilitate  the  detection  of  infec- 
tion. This  concentration  is  accomplished  by  increasing  the  time 
spent  on  the  treatment  of  the  feces  previous  to  their  microscopic  ex- 


PROPERTIES   USED    IX    CONCENTRATION.  13 

amination.  At  the  same  time  it  increases  the  likelihood  of  lindinjj; 
parasites  and  their  eg<j;s  when  these  are  present. 

The  justification  for  thus  .shiftin*^  the  greater  amount  of  time  from 
that  of  microscopic  examination  to  that  of  preliminary  treatment  is 
readily  found.  Microscopic  examination  of  feces  for  parasites  and 
their  eggs  is  one  which  calls  for  some  degree  of  mental  concentration. 
The  judgment  is  constantly  exercised  in  passing  upon  various  objects 
which  catch  the  eye  and  by  a  superficial  resemblance  in  size,  shape, 
color  or  refraction  to  parasites  and  eggs  call  for  a  determiiuition  of 
their  spurious  character.  The  determination  of  some  of  these  forms 
requires  the  use  of  the  higher  powers  of  the  microscoj)e  and  some 
little  time  in  examination.  In  addition  to  the  fact  that  microscopic 
examination  makes  considerable  mental  demands,  is  the  fact  that 
microscopic  work  is  eyestrain — a  matter  of  considerable  importance 
to  anyone  who  has  much  work  of  the  kind  to  do — and  the  saving  of 
eyestrain  by  eliminating  part  of  the  objects  which  must  be  picked  up 
by  the  eye,  only  to  be  rejected  as  not  being  the  thing  sought  for,  is 
greatly  to  be  desired. 

On  the  other  hand,  the  treatment  of  feces  preliminary  to  micro- 
scopic examination  calls  for  little  mental  concentration  and  no  eye- 
strain, and  so  leaves  the  mind  free  for  other  things  and  piolongs  the 
period  of  usefulness  of  the  eyes.  Moreover,  the  fact  that  this  pre- 
liminary treatment  can  largely  be  left  to  the  operation  of  natural 
laws  and  to  power  machinery  also  saves  to  the  operator  the  time  when 
things  can  be  left  unattended. 

PROPERTIES  ISED  IX  COXCEXTRATIOX. 

Concentration,  the  object  of  all  improvements  on  the  smear  method, 
is  obtained  by  eliminating  as  nmch  as  possible  of  the  nonparasitic 
material  by  taking  advantage  of  difi'erences  in  physical,  chemical,  and 
biological  properties  between  such  material  and  the  parasites.  These 
differences  are  in  most  cases  those  of  specific  gravity,  of  size,  of 
physical  and  chemical  solubility,  of  adhesiveness,  and  of  capacity  for 
growth  and  development. 

IMIYSICAI.   PROI'KRTIIS. 

The  elimination  of  matter  having  a  specific  gravity  less  than  that 
of  the  parasites  is  accomplished  by  sedimenting  in  water  and  decant- 
ing material  which  floats  or  remains  sus})ended.  and  by  centrifuging 
in  water,  and  is  based  on  the  general  truth  that  parasites  are  heavier 
than  water  and  will  settle  to  the  bottom.  Doi-k  and  Bass  (1910)  lay 
emphasis  on  the  fact  that — 

Most  ♦  *  *  food  particles  arc  irreuular  in  outline  and  sliajie.  making 
their  surface  l>er  giveu  weight  greater  than  that  of  the  round,  oval  eggs. 


14  METHODS   OF   EXAMINING   FECES   FOR   PARASITISM. 

Owing  to  the  greater  resistance  offered  by  such  surfaces,  these 
particles  are  slower  in  reaching  the  bottom. 

In  Bass's  (1909)  second  method  additional  matter  of  specific 
gravity  less  than  that  of  the  parasite  is  eliminated  by  the  use  of  a 
calcium-chlorid  solution  having  a  specific  gravity  of  1,050.  In  this 
method  and  in  Bass's  (1906)  first  method  matter  having  a  specific 
gravity  greater  than  that  of  the  parasites  is  also  eliminated,  in  the 
first  method  by  the  use  of  a  nine-tenths  saturated  solution  of  sodium 
chlorid  and  in  the  second  method  by  the  use  of  a  calcium-chlorid  solu- 
tion having  a  specific  gravity  of  1.250,  Garrison  (1910)  varies  this 
method  by  using  only  one  solution,  of  specific  gravity  1.200,  and 
Wellman  substitutes  sodium  acetate  for  calcium  chlorid. 

These  methods  and  the  sedimentation  and  filtration  methods  like- 
wise eliminate  matter  which  is  soluble  in  water.  Washing  not  only 
assists  in  concentrating  material  by  removing  fine  suspended  and 
colored  soluble  matter,  but  in  so  doing  it  gives  a  clearer  background 
for  microscopic  work. 

The  elimination  of  matter  larger  than  the  parasites  is  accomplished 
by  the  use  of  sieves  with  a  mesh  aperture  of  such  size  that  it  will  per- 
mit the  passage  of  parasitic  material,  but  not  of  coarse  fecal  particles. 

The  elimination  of  matter  having  different  adhesive  properties,  as 
advocated  by  Pepper  (1908),  has  only  a  limited  application  and 
needs  no  discussion  in  a  consideration  of  general  methods.  The 
writer  has  tested  this  method  and  finds  it  very  satisfactory  for  the 
purpose  for  which  it  was  proposed.  Dock  and  Bass  (1910)  commend 
Pepper's  method,  and  state  in  comment : 

The  best  results  are  obtained  if  a  part  of  the  fecal  material  is  removed  by  use 
of  the  centrifugal.     Otherwise  the  method  is  often  disappointing. 

Other  methods  of  differentiation,  as  staining  reaction,  etc,  might 
be  devised.  In  fact,  as  later  indicated,  the  writer  has  attempted 
something  of  the  sort  without  obtaining  satisfactory  results.  Never- 
theless satisfactory  methods  might  be  devised. 

CHEMICAL   PROrERTIES. 

The  elimination  of  matter  having  a  different  chemical  solubility 
is  accomplished  by  the  addition  of  chemicals  which  will  dissolve  part 
of  the  nonparasitic  substances  with  the  formation  of  new  and  soluble 
substances.  Such  chemicals  will  also  dissolve  a  number  of  things 
forming  a  mere  physical  solution. 

BIOLOGICAL   PROPERTIES, 

The  elimination  of  the  nonparasitic  matter  lacking  the  biological 
properties  of  growth  and  development  is  accomplished  by  the  use  of 
culture  methods.  These  methods,  which  are  indispensable  in  bac- 
teriology and  of  increasing  importance  in  protozoology,  in  both  of 


THEORETICAL   IDEAL   AND   PRACTICAL   OBJECTIONS.  15 

which  the  reproductive  faciihy  may  he  taken  advajitajro  of,  are  of 
limited  use  in  the  examination  of  feces  for  worm  parasites,  as  mul- 
tiplication of  the  parasites  under  these  circumstances  is  only  pos- 
sihle  in  such  exceptional  cases  as  i^tronyyloaleH. 

THE  THEORETICAIi   n)EAL. 

From  the  foregoin*;  it  would  appear  that  the  hest  technique  should 
eliminate  matter  of  specific  <;ravity  different  from  that  of  the  para- 
sites by  sedimentation,  or,  since  it  is  more  rapid,  by  centrifu^iing,  in 
solutions  of  such  strengths  as  to  get  rid  of  the  lighter  nonparasitic 
material  at  one  time  and  the  heavier  at  another:  such  technique 
should  eliminate  matter  of  different  size  by  sieving;  finally  it  should, 
in  the  course  of  the  above  processes  or  the  filtration  method,  eliminate 
matter  of  physical  or  chemical  solubility  different  from  that  of  the 
parasites  by  the  use  of  suitable  reagents.  Such  a  method  would  be 
expected  to  give  a  slide  with  the  nuiximum  amount  of  parasitic  mate- 
rial and  the  minimum  amount  of  other  material.  Incidentally,  this 
treatment  would  comminute  the  resultant  sediment  so  that  it  would 
give  a  clear,  uniform  microscopic  field,  superior  to  that  obtained  by 
the  smear  method.  An  ordinary  smear  of  feces,  subjected  to  no  pre- 
liminary treatment,  makes  a  very  trashy  and  indifferent  microscopic 
preparation,  especially  in  the  herbivora,  where  large  plant  particles 
are  easily  capable  of  concealing  the  evidences  of  parasitic  infection, 
and  the  .same  objection  holds  good  to  some  extent  of  smear  prepa- 
rations of  any  sort  of  feces. 

PR.\CTIC.\L.  OBJECTIONS. 

In  actual  practice,  experience  modifies  the  view  that  we  should  do 
all  the  above  things  and  shows  that  there  are  other  things  that  must 
be  considered.  Among  these  are  (1)  the  possible  injury  to  parasites 
or  eggs  by  certain  forms  of  technicpie,  and  {'1)  the  possibility  that  in 
general  work  certain  methods  may  ])r()ve  to  have  oidy  a  limited  ap- 
plication, or  the  concentration  resulting  from  the  application  of  cer- 
tain methods  may  be  too  insignificant  to  wanant  the  time  and  eff'ort 
expended.  A  discussion  of  these  i)oints  can  best  be  given  after  con- 
sideration of  the  writer's  method. 

GROSS   EXAMINATION. 

The  naked-eye  examination  of  feces  for  gross  evidences  of  para- 
sites, as  stated  at  the  beginning  of  this  paper,  receives  too  little  atten- 
tion, and  it  may  be  fairly  assumed  from  the  lack  of  statements  show- 
ing a  definite  techni(pie  that  it  usually  consists  of  ])erhaj)s  a  hasty 
glance  at  bottled  material  or  possibly  some  jiicking  over  of  the  feces 
with  a  dissecting  needle  or  a  stirring  rod  at  the  most. 


16  METHODS   OF   EXAMINING   FECES   FOE   PARASITISM. 

The  likelihood  of  finding  easily  identified  mature  worms  or  tape- 
worm segments  warrants  a  more  careful  examination.  Garrison 
(1910)  has  recognized  this  fact  and  given  a  method  of  collection  as 
f  ollow^s : 

In  the  case  of  the  larger  worms,  such  as  Aficaris  and  the  larpe  tapeworms, 
which  are  easily  piclted  ont,  about  the  only  precautions  needed  are  to  clean  the 
worms  of  the  fecal  debris  and  to  l<eep  tlieni  as  fresli  as  possible  until  they  are 
killed.  This  is  best  done  by  transferring  them  promi)tly  from  the  fresh  stool 
to  a  dish  of  warm  physiological  salt  solution.     *     *     * 

Smaller  worms,  like  hookworms,  pinwoi'ms,  whipworms,  and  the  "  dwarf 
tapeworm,"  require  careful  searching,  and  this  is  best  done  by  spreading  por- 
tions of  the  fluid  stool  in  a  thin  translucent  layer  in  a  glass  dish  or  on  a  glass 
plate  (table  top),  the  glass  being  placed  over  a  black  background. 

In  picking  out  the  still  smaller  forms,  such  as  the  adult  trichinae,  the  minute 
intestinal  fluke,  Hetcrophyes,  and  the  detached  heads  of  tapeworms,  a  hand 
lens  is  almost  necessary,  and  it  is  advisable  to  make  a  routine  practice  of  run- 
ning over  the  material  with  a  magnifier  of  low  power  in  every  case. 

Should  the  stool  not  be  sufficiently  fluid  to  admit  of  the  above  manipulation, 
it  must  be  thoroughly  mixed  and  diluted  with  water,  and  this  l)rings  up  the 
method  of  sedimentation,  which,  while  of  special  use  in  collecting  ova,  is 
frequently  a  valuable  help  'n  finding  the  smaller  worms. 

THE   WRITER'S  METHOD. 

The  writer's  method  includes  both  macroscopic  and  microscopic 
examination.  Concentration  is  attained  by  careful  comminution  of 
feces,  the  use  of  sieves,  sedimentation  and  centrifuging,  and  washing 
in  water.  The  result  is  checked  by  centrifuging  one  tube  of  ma- 
terial in  a  calcium  chlorid  solution  with  a  specific  gravity  of  1.250, 

The  illustration  (fig.  1)  is  intended  to  show  at  a  glance  the  appa- 
ratus needed  (except  for  pipettes  and  brushes)  and  the  method  and 
order  of  its  use.  It  is,  of  course,  evident  that  the  reader  will  be 
familiar  with  the  individual  pieces  of  apparatus, 

COMMINUTION    OF   FECES. 

In  any  method  which  aims  at  a  concentration  of  the  parasitic  ma- 
terial present  it  is  first  necessary  to  break  up  the  fecal  masses  in 
order  to  release  the  embedded  and  adherent  parasites  and  to  put 
the  feces  in  such  shape  that  they  can  be  treated  by  any  of  the  proc- 
esses outlined  in  the  preceding  part  of  the  paper.  This  is  a  feature 
to  which  it  seems  that  too  little  attention  is  paid.  Of  the  methods 
of  comminuting  such  fecal  masses,  boiling  in  water,  as  advocated 
by  Cobb  (1904)  for  sheep  feces,  is  fairly  good,  but  not  so  effective 
for  sheep  feces  as  the  general  method  used  by  the  writer,  and  has 
obvious  objections  in  the  cases  of  human  feces  or  those  of  the  carniv- 
ora.  Stiles  (1902hh,  19031)  states  that  the  feces  should  be  shaken 
or  stirred  thoroughly,  and  Telemann    (1908)    notes  that  the   feces 


Fia.  1.— Apparatus  used  in  the  writer's  method  of  examining  feces:  a,  Shaker;  b.  brass  sieves  and 
dish:  c,  silk  sieve  and  jar;  d.  beaker:  c,  centrifuge;  /,  micrc^cope. 

78939°— Bull.  135—11 3 


18  METHODS   OP   EXAMINING   FECES   FOR  PARASITISM. 

should  be  shaken  in  the  mixture  of  ether  and  hydrochloric  acid. 
Garrison  (1910)  advises  the  use  of  large  quantities  of  water,  5  to  10 
liters,  for  the  first  sedimentation  of  large  solid  stools,  the  water  to 
be  run  in  violently  and  the  mixture  well  stirred. 

The  writer  takes  particular  care  to  break  up  the  feces  thoroughly. 
The  method  consists  in  shaking  the  feces  in  a  rubber-stoppered,  wide- 
mouthed  glass  bottle  about  three-fourths  full  of  water.  The  entire 
fecal  sample,  up  to  4  or  5  ounces,  is  used.  It  is  sometimes  desirable 
to  break  or  crush  with  a  stirring  rod  such  hard  fecal  masses  as  sheep 
feces.  It  is  also  sometimes  desirable  to  add  shot  to  hard  fecal  masses. 
In  such  cases  the  most  satisfactory  results  were  obtained  with  about 
100  lead  shot  having  a  diameter  of  3.8  millimeters;  shot  with  a 
diameter  of  8  millimeters  was  not  as  effective  in  breaking  up  feces, 
and  had  the  additional  disadvantage  that  it  blackened  the  glass. 
The  use  of  shot  is  to  be  avoided  as  a  rule,  for  the  reason  that  gross 
parasitic  material  is  apt  to  be  damaged  by  it.  At  first  the  bottle 
containing  the  feces  and  water  and,  if  necessary,  the  shot,  was  shaken 
rapidly  by  hand,  but  as  the  amount  of  fecal  examination  in  this 
laboratory  warranted  the  use  of  a  machine  for  this  work,  a  shaker 
of  the  kind  used  in  mixing  "  milk  shakes  "  was  installed  and  con- 
nected by  belting  with  a  pulley  wheel  fitted  on  an  electric-fan  motor. 
This  apparatus  (fig.  1,  a)  is  very  rapid  and  effective  in  its  work. 
The  bottles  are  lifted  a  distance  of  5  centimeters  and  dropped  back 
again  at  a  rate  of  about  500  times  per  minute.  The  same  machine 
operated  by  hand  would  doubtless  be  very  good. 

SIEVING. 

After  having  been  broken  up  in  this  manner  the  feces  are  poured 
through  a  set  of  six  brass  sieves.  The  sieves  have  a  mesh  aperture 
ranging  from  3  millimeters  in  the  largest  to  about  one-fourth  of  a 
millimeter  in  the  smallest.  They  are  made  by  taking  tin  pans  with 
a  bottom  diameter  of  about  6|  inches  and  sides  2  inches  high,  cutting 
out  the  bottom,  leaving  a  flange  near  the  sides,  and  soldering  onto  the 
flange  brass  screening  with  meshes  of  various  sizes.  These  sieves 
are  copied  from  a  set  used  by  Dr.  Cobb  in  collecting  free-living 
nematodes.  The  pans,  of  course,  tend  to  rust,  as  it  is  not  always 
convenient  to  dry  them  after  use.  Dr.  Cobb  tells  me  that  he  avoids 
this  by  the  use  of  oil  or  grease  warmed  on  the  pans  and  then  care- 
fully wiped  off.  As  this  practice  seems  inconvenient  also,  the  pans 
used  by  the  writer  have  been  enameled  to  prevent  rusting.  This  makes 
the  pans  bind  a  little  when  nested.  A  set  coated  with  shellac  is  being 
tried.  Galvanized-iron,  brass,  or  aluminum  pans  would  presum- 
ably be  better,  though  the  two  latter  would  be  more  expensive.  The 
brass  sieves  which  can  be  purchased  already  made  are  not  beveled 


SIEVING.  19 

and  hence  can  not  be  nested.  They  can  be  superimposed,  one  on 
another,  but  the  result  is  a  stack  which  is  so  hi<^h  that  it  does  not 
permit  of  six  of  tliem  being  set  in  a  shallow  dish  of  water  with  the 
water  standing  above  the  screen  in  the  upper  sieve. 

The  sieves  are  nested  in  the  order  of  mesh  aperture,  with  the  coars- 
est on  top,  and  placed  in  a  large  porcelain  evaporating  dish,  or  a 
large  glass  crystallization  dish  (fig.  1,  h).  The  feces  are  poured  into 
the  top  screen  and  pass  through  the  screens  to  the  evaporating  dish, 
particles  of  different  sizes  being  held  by  the  different  screens.  Tiie 
use  of  fewer  screens  would  not  be  a  gain,  as  too  coarse  material 
poured  on  a  screen  clogs  it.  Tap  water  or  normal  salt  solution  is 
poured  in  the  upper  sieve  until  the  water  stands  in  the  evaporating 
or  crystallization  dish  at  a  level  above  that  of  the  bottom  of  the 
upper  sieve.  This  sieve  is  lifted  and  shaken  a  little  until  the  fine 
matter  has  passed  through.  It  is  then  lifted  out  and  put  in  a  large 
crystallization  dish  half  full  of  water  or  salt  solution  and  the  matter 
it  contained  examined  on  the  screen  or  washed  into  the  dish.  Groses 
parasitic  material  is  picked  out,  the  screen  rinsed,  the  dish  emptied 
and  refilled  if  the  amount  of  discoloration  or  trash  present  war- 
rants it,  and  the  process  is  repeated  with  the  remaining  sieves.  The 
gross  material  left  on  the  screens  is  thoroughly  cleaned,  and  the  like- 
lihood of  wasting  time  examining  citrus  pulp  vesicles,  vegetable  fibers, 
etc.,  as  possible  parasites  is  reduced  to  a  minimum. 

The  sediment  left  in  the  evaporating  dish  after  removing  the  finest 
sieve  is  poured  onto  a  screen  of  miller's  silk  bolting  cloth  with  a  mesh 
aperture  of  0.117  to  0.134  millimeter  and  the  finer  particles  washed 
through  into  a  tall  jar  (fig.  1,  c).  The  mesh  aperture  of  this  sieve 
diminishes  as  the  cloth  shrinks  with  use,  and  in  a  cloth  which  has  been 
in  use  for  several  months  it  has  diminished  to  a  size  of  0.070  to  0.080 
millimeter.  Such  a  mesh  is  too  fine  to  permit  the  passage  of  the  eggs 
of  such  important  species  as  Fasciola  hcpatUn.  Some  No.  120  mesh 
brass  screen  has  recently  been  obtained  which  has  a  mesh  aperture 
rated  at  0.117  millimeter,  but  varving  from  0.103  to  0.120,  accordinir 
to  the  writer's  measurements.  This  promises  to  be  a  very  satisfactory 
substitute  for  the  bolting  cloth.  Presumably  it  will  retain  its  mesli 
aperture,  and  will  be  more  durable. 

When  the  shot  are  used,  they  are  poured  with  the  feces  into  the 
coarsest  of  the  brass  sieves.  They  are  subsequently  poured  from  the 
sieve  into  a  petri  dish  with  whatever  coarse  fecal  matter  may  accom- 
pany them  and  the  fecal  matter  easily  removed  by  a  stream  of  tap 
water.  It  might  be  supposed  that  fecal  matter  of  some  sort  would 
stick  to  the  shot,  but  it  does  not  do  so.  Parasitic  material  that  might 
adhere  can  be  destroyed  by  dry  heat.  Lead  shot  have  the  advantage 
over  steel  shot  that  they  may  be  kept  in  a  formol  ^olution  without 
rusting,  while  steel  shot  would  need  careful  drying,  or  else  keeping 
in  oil. 


20  METHODS    OF    EXAMINING    FECES    FOR    PARASITISM. 

Where  small  amounts  of  feces  are  used,  two  pieces  of  brass  tubings 
2  inches  in  diameter  and  1^  inches  high,  beveled  to  fit  one  in  the 
other,  and  the  lower  one  furnished  with  three  projections  to  hold  it 
on  glassware  of  not  over  3  inches  in  diameter,  are  used  to  hold  the 
bolting  cloth  of  the  silk  sieve,  the  cloth  being  caught  and  held  taut 
by  the  beveled  surfaces.  Where  large  amounts  of  feces  are  used,  and 
large  amounts — not  to  exceed  4  or  5  ounces— should  be  used  whenever 
obtainable,  two  enameled  tin  pans,  with  a  bottom  diameter  of  4^ 
inches  and  with  the  bottom  cut  out  so  as  to  leave  a  narrow  flange,  are 
used  in  place  of  the  brass  tubing.  The  cloth  is  held  between  the  upper 
flanges  of  the  tw^o  tins,  and  the  cloth  and  flanges  held  together  by 
four  small  clothespins  of  the  sort  provided  with  a  wdre  spring  to 
hold  the  jaws  together.  This  device  I  also  ow-e  to  Dr.  Cobb.  The 
sieve  formed  of  the  tins  and  the  bolting  cloth  is  the  right  size  for 
use  with  a  jar  10  inches  high  and  5  inches  in  diameter  (see  fig.  1,  c). 
When  necessary  a  soft  brush  is  used  to  brush  the  feces  through  the 
fine  brass  sieves  or  the  silk  cloth.  Cobb  (1904),  as  previously  noted, 
uses  a  brush  for  the  same  purpose.  These  sieves  will  Avork  better  if 
water  is  poured  through  them  or  if  they  are  dipped  in  water  before 
the  fecal  matter  is  jDoured  on. 

The  sieves,  as  well  as  all  other  apparatus  coming  in  contact  with 
the  fecal  material,  are  washed  promptly  with  boiling  water,  the  sieves 
being  also  scrubbed  with  a  stiff  brush.  This  prevents  any  parasitic 
material  from  remaining  to  contaminate  a  subsequent  fecal  specimen, 
and  thereby  giving  inaccurate  findings.  A  microscopic  examination 
of  the  silk  sieve  shows  that  it  washes  clean  very  readily,  and  when 
rinsed  retains  very  little  of  the  material  poured  on  it.  Experiment 
shows  that  eggs  pass  through  this  cloth  very  readily,  less  than  half 
of  1  per  cent  of  even  such  large  eggs  as  Toxocara  remaining  when 
the  fecal  matter  is  first  brushed  through.  A  smear  made  from  the 
residue  on  the  bolting-cloth  sieve  showed  4  eggs  in  one  case  where 
the  slide  preparation  from  the  centrifuged  sediment  showed  860  eggs; 
another  smear  from  the  residue  on  the  bolting  cloth  showed  1  egg 
where  the  slide  from  the  sediment  showed  475  eggs.  In  the  latter 
case,  a  fair  estimate  of  the  amount  of  material  on  the  cloth  and  the 
amount  from  which  the  smear  was  made  indicates  that  there  were 
probably  not  more  than  10  eggs  left  on  the  cloth,  while  thousands 
had  passed  through  it.  Those  that  are  left  are  held  by  the  jelly-like 
residue  obtained  at  this  point,  and  not  because  the  screen  mesh  is 
defective  or  too  small.  The  writer  is  not  aware  of  any  parasite  eggs 
which  are  too  large  in  their  smaller  diameter  to  pass  through  a  mesh 
with  a  diameter  of  0.117  to  0.134  millimeter.  The  number  remain- 
ing after  clean  water  has  been  poured  through  the  cloth  into  the  jar 
is  entirely  negligible. 

In  working  with  human  feces,  or  where  dangerous  infection  may 
be  present,  the  silk  cloth  may  be  kept  in  a  jar  of  formol  solution 


REDTMENTING    AND   CEXTRTFUGTXG,  21 

when  not  in  use.  In  tlie  course  ol"  a  lai<^e  number  of  experinients 
nothing  has  yet  indicated  tliat  parasitic  material  from  one  examina- 
tion has  remained  to  subsequently  contaminate  other  fecal  speci- 
mens. Parasites  that  might  be  suspected  of  remaining  after  the 
cloth  had  been  washed  in  boiling  water  might  be  destroyed  by  pro- 
longed boiling  or  subjection  to  dry  heat — experiment  shows  lliat 
eggs  so  treated  are  distorted  or  characterized  by  the  formation  of 
air  spaces  or  oily  areas — or  fresh  pieces  of  cloth  could  be  used  each 
time.  This  last,  however,  would  be  somewhat  expensixc,  as  this 
cloth  retails  in  Washington  in  half-yard  widtlis  at  about  $.")  a  yard. 
It  would  be  cheaper  to  use  the  Xo.  120  mesh  brass  screen.  This  costs 
$1.85  a  square  foot,  but  would  b(>  jiernuuient. 

SEDIMKNTIXCi    AND    (  ENTRI  Fl CINC 

The  feces  which  pass  through  the  silk  sieve  are  sedimented  with 
plenty  of  water  in  the  jar.  After  decanting,  the  sediment  is  trans- 
ferred to  a  beaker  (tig.  1.  d)  and  may  now  be  washed  if  desired. 
The  entire  sediment,  or  as  much  as  seems  desirable,  is  then  centrifuged 
(see  fig.  1,  e) ,  repeated  centrifuging  with  the  addition  of  fresh  ma- 
terial adding  to  the  total  centrifuge  sediment,  and  may  be  washed 
at  this  point  also,  as  advised  by  Pepper  (1908)  and  Bass  (1909). 
The  writer  sometimes  washes  the  material  at  both  points,  the  second 
supplementing  and  completing  the  first.  It  is  usually  sufficient  to 
wash  the  sediment  in  the  centrifuge.  Bass  has  called  attention  to  the 
important  fact  that  a  centrifuge  should  only  be  run  the  minimum 
time  necessary  to  bring  down  the  eggs.  This  time  will  vary  with 
different  centrifuges.  AVith  a  centrifuge  running  8.500  revolutions 
per  minute  Bass  allows  4  to  10  seconds,  I  find  this  enough  time 
with  a  centrifuge  running  1.230  revolutions  per  minutes.  After  the 
material  in  the  two  centrifuge  tubes  is  washed  in  water,  one  tube  is 
left  alone;  the  water  is  poured  off  the  other  and  calcium  chlorid  solu- 
tion, with  a  specific  gravity  of  1,250,  is  added  to  the  sediment.  After 
centrifuging,  a  slide  preparation  is  sometimes  made  from  this  tube 
direct.  In  most  cases  the  top  cubic  centimeter  is  jjipetted  off.  shaken 
up  with  14  cubic  centimeters  of  water,  and  ceuti'ifuged.  This  is  the 
more  satisfactory  and  certain  method. 

I'RKI'AKATION    (»K    SIJDKS. 

By  means  of  a  long  pijK'tte,  a  drop  of  sediment  is  drawn  up  from 
the  bottom  of  the  tubi'  in  which  water  alone  is  used,  placed  on  a  slide 
under  a  cover  glass,  and  examined  with  a  microscope  (fig.  1.  /).  A 
second  slide  is  nuide  from  the  other  tube.  This  second  slide  is  cither 
made  directly  from  a  drop  taken  from  the  surface  of  the  calcium 
chlorid  solution,  or  from  the  bottom  in  case  the  top  cubic  centiuieter 


22  METHODS   OF   EXAMINING   FECES   FOR   PARASITISM. 

has  been  added  to  water  and  centrifuged.  The  second  slide  is  used 
as  a  check  on  the  first.  It  sometimes  has  fewer  eggs,  especially  when 
pipetted  direct  from  the  top,  but  it  is  a  cleaner  preparation,  is  easily 
examined,  will  sometimes  have  more  eggs,  especially  if  made  from  the 
sediment  where  the  top  cubic  centimeter  of  the  1.250  solution  has 
been  centrifuged  Avith  the  addition  of  water,  and  occasionally  throws 
additional  light  on  the  material  under  examination.  The  pipettes 
are  rinsed  thoroughly,  and  when  dried  are  heated  in  a  Bunsen  flame 
for  a  short  time  to  destroy  any  eggs  that  might  adhere,  thus  prevent- 
ing contamination  in  subsequent  examinations. 

CONCENTRATION   OBTAINED  BY  THE  USE  OF  SIEVES. 

In  examining  the  feces  of  35  sheep,  the  entire  amount  of  feces  pass- 
ing through  the  sieve  was  centrifuged  in  order  to  give  a  uniform 
comparative  study  and  to  determine  the  amount  of  concentration 
attained  by  the  use  of  sieves,  and  due  to  them  alone.  To  eliminate 
other  factors,  the  sediment  was  not  washed  and  the  centrifuge  was 
run  for  long  periods  till  everything  had  come  down.  Centrifuging 
the  entire  amount  of  feces  necessitated  the  repeated  filling  of  the 
tubes  of  a  two  or  four  arm  centrifuge.  A  comparative  examination 
of  slides  made  from  the  sediment  obtained  by  centrifuging  a  single 
tube  full  of  the  material,  with  slides  made  from  the  total  sediment, 
showed  that  the  concentration  was  the  same  in  both  cases,  a  result 
which  would  be  expected  from  a  theoretical  standpoint.  While  the 
concentration  is  the  same,  the  total  amount  of  parasitic  material 
present  is,  of  course,  much  less  in  the  single  tube. 

Using  moist  fecal  pellets,  the  concentration  obtained  was  4 : 1.  The 
concentration  varies  with  animals  of  other  species,  with  food  habits, 
and  with  the  condition  of  the  particular  fecal  specimen  examined. 
At  the  same  time,  the  concentration  is  always  sufficient  to  warrant 
the  use  of  the  sieves.  The  microscopic  field  obtained  after  treatment 
of  feces  in  this  way  is  very  much  more  satisfactory  than  the  field 
obtained  in  the  smear  method,  and  where  the  same  number  of  slides 
are  examined  the  likelihood  of  finding  evidences  of  existing  parasit- 
ism is  certainly  more  than  four  times  greater  in  cases  where  the  feces 
have  been  subjected  to  thorough  sieving. 

SUMMARY   OF   METHOD. 

The  writer's  method  is,  then,  merely  a  modification  of  existing 
methods,  and  might  be  termed  a  comminution-sieving-sedimentation- 
centrifuge  method  in  which  water  alone  is  depended  on  as  a  medium 
for  these  operations,  a  slide  made  after  centrifuging  in  a  calcium 
chlorid  solution  with  a  specific  gravity  of  1.250  being  regarded  prin- 
cipally as  a  check  on  the  method  as  given. 


COLLECTION    INDEX.  23 

COLLECTION    INDEX. 

The  writer  has  had  no  opportunity  to  work  out  an  index  showing 
the  rehition  of  the  count  obtained  by  the  writer's  method  to  the  num- 
ber of  worms  present.  In  few  cases  where  the  feces  of  animals  are 
examined  in  this  hiboratory  are  the  animals  examined  post-mortem 
within  a  short  time.  In  one  instance.  3  grams  of  feces  were  taken 
from  the  rectum  of  a  sheep  during  a  post-mortem  examination  for 
parasites,  and  after  treatment  by  the  writer's  method,  noting  the  time 
element  and  the  amount  of  water  used  in  each  case — which  data  need 
not  be  given  here — a  slide  was  made  of  one-tenth  of  a  cubic  centi- 
meter of  sediment.  This  slide  showed  3,325  nematode  eggs  and  em- 
bryos. The  intestines  and  fourth  stomach  showed  a  correspondingly 
high  degree  of  infection  with  numerous  species  of  worms,  and  it  was 
impossible  to  determine  the  relation  between  the  number  of  female 
w^orms  of  any  species  and  the  number  of  eggs  of  that  species  under 
the  circumstances. 

In  spite  of  the  relation  found  by  Cobb  (1004)  as  regards  flukes. 
by  Leichtenstern  and  by  Grassi  and  Parona,  according  to  Dock  and 
Bass  (1910),  as  regards  hookworms,  between  the  number  of  parasites 
present  and  the  number  of  eggs  in  a  given  fecal  sample,  the  examina- 
tions made  by  the  writer  do  not  indicate  that  anything  of  the  sort 
is  apt  to  prove  of  use  in  routine  examinations  for  parasites  in  general. 
Dock  and  Bass  (1910)  have  come  to  similar  conclusions  and  cite  the 
work  of  Ashford  and  King  to  the  same  effect.  Not  only  is  there 
a  certain  element  of  chance  in  the  detection  of  eggs  when  they  are 
present  in  light  infections,  but  there  are  certain  conditions  which 
permit  of  the  existence  of  parasitism  without  the  presence  of  eggs 
in  the  feces  to  indicate  it.    Some  of  these  conditions  are  as  follows: 

(1)  Infection  with  male  nematodes  will  not  be  indicated  by  eggs 
in  the  feces.  Occasionally  the  only  nematodes  of  a  given  species 
which  are  present  in  an  animal  will  be  males.  Such  infections  are 
usually  light. 

(2)  The  above  condition  will  not  be  true  of  the  flukes  and  tape- 
worms, as  they  are  hermaphroditic;  but  in  the  case  of  these  worms, 
and  the  nematodes  also,  infections  which  are  so  recent  that  the 
infecting  larvae  have  not  yet  reached  the  egg-producing  stage  will 
not  be  indicated  by  eggs  in  the  feces.    Such  infections  may  be  heavy. 

(3)  ?]gg  production  in  female  or  hermaphroditic  animals  may  be 
interrupted  in  various  ways.  Thus  tapeworms  may  be  broken,  per- 
haps by  intestinal  peristalsis,  at  points  anywhere  from  just  back  of 
the  head  to  just  in  front  of  the  gravid  proglottids.  and  the  feces  will 
show  no  more  eggs  or  proglottids  until  new  gravid  proglottids  are 
formed.  Dock  and  Bass  (1910)  report  that  in  an  examination  of 
247  female  Nccator  at/icricatm^  by  Bass,  7  per  cent  were  found  to 


24  METHODS   OF   EXAMINING   FECES   FOR   PARASITISM. 

contain  no  ova.  Bass  suggested  the  possibility  of  old  age  as  an 
explanation  of  this.  Dock  and  Bass  (1910)  state  that  they  found 
9.25  per  cent  of  397  female  N.  americanus  barren  in  cases  Avhere  the 
infection  was  of  long  standing,  while  only  one-third  of  1  per  cent 
of  120  female  hookworms  from  the  dog  were  without  eggs.  They 
state,  however,  that  Dr.  Stiles  does  not  think  that  hookworms  live 
long  after  old  age  induces  cessation  of  ovulation. 

(4)  A  thorough  purging  of  the  intestinal  tract  may  remove  an 
accumulation  of  parasite  eggs,  with  the  result  that  numbers  of  eggs 
will  be  found  at  this  time  and  none  a  day  or  two  later.  Dock  and 
Bass  (1910)  comment  favorably  on  Ashford  and  King's  statement 
that  hookworms  cause  an  increase  of  mucus  at  the  site  of  their  feeding 
ground,  and  as  this  mucus  often  comes  off  en  masse  and  contains 
most  of  the  eggs  the  actual  feces  would  contain  relatively  few.  They 
also  note  that  Ashford  and  King  say  that  eggs  are  more  difficult  to 
find  in  diarrheal  stools,  and  that  they  saw  cases  of  heavy  infection 
that  at  times  had  no  ova  in  the  feces.  Dock  and  Bass  (1910)  call 
attention  to  the  fact  that  the  amount  of  feces  varies  with  different 
persons  and  from  time  to  time  in  the  same  person.  They  also  state 
that  it  is  a  common  experience  to  find  no  eggs  in  the  feces  within  a 
few  days  after  a  course  of  thymol  and  subsequently  to  find  many 
eggs.  They  say  that  Dieminger  found  that  the  number  of  eggs  was 
very  much  diminished  when  patients  were  drinking  hard. 

It  follows  from  the  above  that  little  weight  can  be  laid  on  a  col- 
lection index.  In  the  Avriter's  experience,  consecutive  and  careful 
examinations  of  the  feces  of  a  given  animal  show  days  when  eggs 
of  various  kinds  are  abundant  and  days  when  they  are  scarce  or 
missing,  and  the  collection  index  for  the  various  days  would  present 
a  striking  disagreement.    This  would  be  particularly  true  of  cestodes. 

Negative  examinations  must  be  considered  doubtful  and  must  be 
checked  at  intervals  if  infection  is  suspected. 

TESTS    OF   UNPUBLISHED   METHODS. 

Numerous  tests  of  unpublished  modifications  of  technique  were 
made  in  connection  with  the  development  of  the  writer's  method. 
Among  these  was  an  attempt  to  increase  the  specific  gravity  of  para- 
sites and  their  eggs  by  adding  mercuric  chlorid  before  centrifuging. 
Comparative  tests  did  not  indicate  that  this  method  was  of  value,  and 
any  adaptation  of  technique  which  would  make  it  so  would  probably 
not  repay  in  results  what  it  required  in  time.  Tentative  attempts  to 
use  a  differential  stain  were  unsatisfactory.  In  one  of  these,  sul- 
phuric acid  and  iodin  were  added  to  slide  preparations  to  distinguish 
cellulose  substances  which  might  otherwise  have  to  be  examined  as 


COMPARATIVE   TESTS   OF    METHODS.  25 

possibly  parasitic.  By  this  niothotl  cellulose  was  colored  violet  or 
black,  and  nematode  eggs  brown,  or  at  times  black,  with  a  light 
areole  where  the  shell  showed  at  the  periphery.  As  such  a  method 
lessened  the  refractive  index  wdiich  makes  most  nematode  eggs  so 
conspicuous,  the  use  of  these  reagents  was  discontimied  as  giving  no 
improvement  in  the  resulting  preparations.  Stubbendorff'  (181)3a) 
has  noted  this  test  and  a  number  of  others  in  a  note  on  the  ditt'erential 
diagnosis  of  parasite  eggs  and  plant  spores. 

COMPARATIVE    TESTS    OF    THE    WRITER'S    METHOD    AND    OTHER 

METHODS. 

For  about  a  j-ear  comparative  tests  of  the  writer's  method  and  other 
methods  were  made  in  the  examination  of  feces  of  various  kinds.  So 
far  as  possible  equal  amounts  of  feces  were  examined  and  the  slides 
made  from  equal  amounts  of  material.  The  ])arasite  eggs  an<l  em- 
bryos were  carefully  counted  with  the  aid  of  a  mechanical  stage,  and 
the  results  compared. 

SMEAK    METHOD. 

Comparative  tests  of  the  smear  method  and  the  writer's  method, 
some  results  of  which  are  given  in  connection  Avith  other  tests,  indi- 
cate that  the  smear  method  is  much  less  certain  and  effective.  A 
diagnosis  made  by  the  writer's  method  would  be  much  more  complete 
and  adequate  than  one  based  on  the  usual  ten  smear  preparations. 

SEDI MENTATION    METHOD. 

The  simple  sedimentation  method  is  so  obviously  inferior  to 
methods  involving  sieving  and  centrifuging,  and  improved  modifica- 
tions of  the  sedimentation  method  were  found  so  inferior  to  the 
writer's  method  in  actual  test,  that  the  simple  method  was  not  even 
given  a  comparative  test. 

151  KPHTE    MprniOD. 

A  test  of  the  sedimentation  method  in  which  the  sediment  was 
taken  from  the  bottom  by  means  of  a  stopcock  did  not  give  as  good 
results  as  the  centrifuge  method.  In  this  test  a  burette  of  '2~)  cubic 
centimeters  capacity  was  used.  Ten  cubic  centimeters  of  water  were 
first  put  in  it,  with  the  idea  of  washing  the  feces  at  the  bottom  of 
the  burette  instead  of  at  the  top,  as  is  commonly  done  where  water 
is  poured  over  a  sediment  and  the  sediment  shaken  up  in  the  watei-, 
allowed  to  settle,  and  the  fluid  then  dtx'anted.  It  appeared  that  in 
this  way  the  sediment  coidd  be  taken  out  promptly  and  already 
washed.     In  testing,  the  sediment  obtained  after  screening  and  de- 


26  METHODS   OF   EXAMINING   FECES   FOB  PARASITISM. 

canting  was  shaken  vigorously  and  divided  into  two  equal  parts,  one 
being  centrifuged  and  washed,  the  other  being  poured  onto  the  10 
cubic  centimeters  of  water  in  the  burette.  As  the  sediment  settled 
it  could  be  traced  by  the  discoloration  of  the  water,  and  when  the 
discoloration  reached  the  bottom  one  preparation  was  made  by  open- 
ing the  stopcock  and  taking  a  drop  of  water  on  the  slide.  Other 
slides  were  made  at  intervals  up  to  an  hour.  In  one  specimen  of  dog 
feces  where  there  was  an  infection  Avith  Dipylidium^  Ascarls,  Tn- 
chuns,  and  Ancylostoma,  the  best  of  six  slides  made  in  this  way 
showed  a  total  of  27  eggs,  while  a  slide  made  after  centrifuging  with 
calcium  chlorid  of  1.250  specific  gravity  showed  77  eggs  and  one 
centrifuged  in  water  alone  showed  181  eggs.  In  the  last  case  there 
were  more  eggs  of  any  one  of  the  four  species  of  parasites  present 
than  there  were  of  all  together  in  the  best  of  the  slides  made  by  the 
burette  method. 

In  another  test,  feces  from  a  dog  infected  with  Toxocara  and  Taenia 
w^ere  tested  by  the  burette  method  without  the  clean  water  at  the  bot- 
tom, the  sediment  obtained  after  screening  being  poured  into  the 
empty  burette.  The  best  of  two  slides  made  by  this  method  showed 
58  eggs,  a  slide  made  by  the  smear  method  showed  46  eggs,  a  slide 
made  by  centrifuging  in  the  solution  of  1,250  specific  gravity  showed 
117  eggs,  and  a  slide  made  after  centrifuging  in  Avater  alone  showed 
215  eggs  in  one- fourth  of  the  cover-glass  area.  The  slide  started  to 
dry  at  this  point  and  the  count  Avas  discontinued.  The  estimated 
total  for  the  slide  Avas  of  course  four  times  215,  or  860  eggs.  A 
smear  preparation  made  from  the  residue  on  the  bolting-cloth  sieve 
shoAved  four  Toxocara  and  no  Taenia  eggs. 

GASTEIGER'S   filter    METHOD. 

A  test  of  the  filter  paper  and  funnel  as  a  substitute  for  the  centri- 
fuge indicated  at  once  that  nothing  was  gained  by  scattering  a  sedi- 
ment over  a  filter  paper  instead  of  concentrating  it  in  the  bottom  of 
a  tube.  The  method  was  also  slow  and  offered  the  usual  chance  of  a 
break  in  the  filter  paper,  necessitating  a  second  filtration.  I  can  not 
imagine  any  use  of  the  filter  in  fecal  examinations  for  parasites  that 
would  not  be  better  subserA^ed  by  the  use  of  the  sieve  followed  by 
centrifuging. 

BASS''S  CALCIUM  CHLORID  CENTRIFUGE  METHOD. 

Inasmuch  as  Bass's  salt  solution  method  is  held  by  its  author  to 
be  inferior  to  the  calcium  chlorid  method,  it  need  only  be  noted  here 
that  in  the  few  tests  of  the  first-named  method  made  by  the  Avriter 


COMPARATIVE   TESTS   OF    METHODS.  27 

its  inferiority  to  the  calcium  chlorid   method   and  to  the  wiiti'i'.s 
method  was  quite  apparent. 

In  comparative  tests  of  Bass's  calcium  chlorid  method,  extending 
over  a  number  of  months  and  involvin<5  the  examination  of  the  feces 
of  man,  the  dog,  sheep,  eland,  hartbeest,  and  chicken,  it  was  found 
that  this  method  secures  a  high  degree  of  concentration  of  material, 
but  in  eliminating  nonparasitic  material  it  also  eliminates  some  para- 
sites. Part  of  these  seem  to  be  left  in  the  rejected  Hnliment  at  the 
bottom  of  the  tube  containing  the  solution  with  a  specific  gravity  of 
l.*250,  as  repeated  examinations  of  this  rejected  sediment  showed  num- 
bers of  parasite  eggs  and  embryos  in  nearly  all  cases,  indicating  that 
endosmosis  brings  the  specific  gravity  of  the  parasites  up  to  that  of 
the  surrounding  fluid.  Some  parasites  seem  to  be  lost  also  in  the 
repeated  handling  due  to  the  use  of  the  solution  with  a  specific  grav- 
ity of  1.050,  as  more  parasites  were  recovered  where  the  use  of  this 
solution  was  omitted  and  the  1.250  solution  alone  used  than  where 
both  were  used,  as  Bass  directs.  Where  slide  preparations  were  made 
from  equal  amounts  of  sediment  secured  b}'  Bass's  method  and  the 
writer's  method,  the  amount  of  parasitic  material  to  a  slide  was  usu- 
ally greater  when  prepared  by  the  writer's  method.  There  is.  fur- 
thermore, a  distortion  of  parasite  eggs  and  embryos  due  to  osmosis. 
This  distortion  and  destruction  of  parasites  constitutes  at  once  a  limi- 
tation and  a  defect  of  the  method.  It  renders  it  unsuitable  for  the 
collection  of  live  material  and  makes  it  more  difficult  to  recognize  and 
identify  some  parasites,  such  as  nematode  embryos. 

In  an  examination  of  some  human  feces  from  a  case  of  hookworm 
infection,  which  material  was  obtained  through  the  courtesy  of  Dr. 
Stiles  and  his  assistant.  Mr.  Crane,  a  smear  preparation  of  the  feces 
showed  2  eggs,  a  preparation  made  by  Bass's  method  showed  84 
eggs,  and  a  preparation  made  by  the  writer's  method  showed  348 
eggs.  An  explanation  of  the  failure  of  Bass's  method  to  give  as 
good  results  was  found  in  an  examination  of  the  sediment  in  the  tube 
containing  the  strong  calcium  chlorid  solution.  A  preparation  made 
from  this  sediment  showed  122  eggs. 

In  an  examination  of  the  feces  of  a  number  of  dogs,  the  feces  were 
treated  according  to  the  method  given  by  the  writer,  according  to 
that  of  Bass,  and  according  to  Bass's  method  without  the  use  of  his 
solution  of  1.050  specific  gravity.  The  method  involving  the  use  of 
the  1.250  solution,  but  not  the  1.050  solution,  is  given  in  the  table 
below  as  Bass's  modified  method.  The  slides  were  made  by  drawing 
up  definite  equal  amounts  of  sediment  in  a  graduated  pipette  from 
the  bottom  of  the  centrifuge  tube.  The  resuHs  of  the  tests  are  given 
in  the  following  table,     (leneric  names  refer  to  eggs  found. 


28  METHODS   OF   EXAMINING   FECES   FOR   PARASITISM. 

Comparative  tests  of  three  methods  in  examination  of  dog  feces. 


Method. 

Writer's. 

Bass's. 

Bass's  modified. 

Dog  No.  1 

Eggs. 
|1  Nematode  embryo. . . 

\8  Ancylostoma 

f28  A ncylostoma 

\2S  Trichuris 

Eggs. 

Eggs. 
Negative. 
23  Ancylostoma. 

Dog  No.  2 

5  Ancylostoma 

18  Trichuris 

3  Trichuris. 

DogNo.3 

Negative. 

16  Ancylostoma. 

Dog  No.  4 

13  Ancylostoma 

20  Ancylostoma 

flO  Ancylostoma 

1  Toxocara. 

/3  Tsenia 

\36  Ancylostoma. 
2  Toxocara. 

Dog  No.  6 

\10  Ancylostoma 

Dog  No.  7 

258  Txnia 

209  Txnia 

299  Txnia. 

An  examination  of  the  above  table  shows  that  the  results  obtained 
by  the  use  of  the  writer's  method  and  of  Bass's  method  without  the 
step  involving  the  use  of  the  solution  of  1.050  specific  gravity,  are 
on  the  whole  superior  to  those  obtained  by  the  use  of  Bass's  method. 
Dock  and  Bass  (1910)  state  of  Bass's  method: 

Gage  and  Bass  conclude,  after  this  extensive  experience  [tlie  examination  of 
the  feces  of  315  students  by  the  smear  method,  by  sedimentation  and  centrifug- 
ing,  and  by  Bass's  method],  that  for  all  practical  purposes  washing  with  water 
alone  is  all  that  is  necessary  and  that  the  washing  with  calcium-chlorid 
solution  is  unnecessary  except  for  special  purposes. 

The  fact  that  the  use  of  the  strong  calcium  chlorid  solution  gives 
at  times  better  results  than  the  use  of  Avater  alone  is  the  writer's 
Avarrant  for  making  use  of  it  in  one  centrifuge  tube  to  check  the 
findings  from  the  other  tube. 

garrison's    calcium    CHLORID    SEDIMENTATION    METHOD. 

Garrison's  method  of  using  calcium  chlorid  and  sedimenting  instead 
of  centrifuging  has  come  to  the  writer's  attention  too  recently  to 
permit  of  adequate  tests.  A  test  made  by  sedimenting  in  a  centrifuge 
tube  instead  of  centrifuging  did  not  give  as  good  results  as  cen- 
trifuging. Sedimenting  has  the  disadvantage  of  being  slower  than 
centrifuging. 

TELEMANN's   ETHER   HYDROCHLORIC   ACID   METHOD. 

A  number  of  tests  were  made  to  determine  the  applicability  of 
Telemann's  (1908)  chemical  methods  to  the  examination  of  feces. 
Telemann  himself  .states  that  he  has  found  his  method  satisfactory  in 
a  large  series  of  human  and  animal  feces.  He  does  not  specify 
what  animals  are  included  in  the  series,  but  his  statement  may  be 
believed  in  any  case.  At  the  same  time,  the  tests  made  in  this 
laboratory  indicate  that  his  success  was  due  more  to  the  use  of  the 
hair  sieve  than  to  the  use  of  chemicals.     The  latter  probably  did 


COMPARATIVE   TKSTS   OF    MF.TIIODS.  29 

more  in  mechanically  breakin<i-  up  the  feces  bv  a  sli*>ht  amount  of 
chemical  action  than  in  actually  concentrating  parasiiic  material 
by  eliminatin*r  nonparasitic  matter  soluble  in  ether  and  hydrochloiic 
acid.  As  previously  stated,  the  tests  of  the  writer's  method  on  sheep 
feces  gave  a  constant  theoretical  concentration  of  4:  1.  The  addition 
of  ether  and  hydrochloric  acid  to  a  given  O..')  cubic  centimeter  of 
sediment  so  obtained  gave  a  reduction  to  0.4  cubic  centimetei',  or  an 
additional  theoretical  concentration  of  only  5:4.  When  the  ether 
and  hydrochloric  acid  were  added  to  the  fresh  fcn-es  a  somewhat 
smaller  sediment  was  obtained.  This  does  not  indicate  a  greater  con- 
centration, as  it  appears  to,  but  a  lesser  breaking  up  of  feces  by  these 
reagents  than  by  water,  so  that  more  agglomerations  of  material 
that  should  be  broken  up  to  allow  the  finer  matter  to  pass  the  sieve 
are  left  unbroken,  and  the  small  as  well  as  the  large  particles  are 
held  by  the  sieve.  This  is  evident  from  the  result  obtained  by  adding 
these  reagents  last  to  determine  the  action  due  to  them  alone. 

Another  result  sometimes  obtained  by  using  Telemann's  method 
on  sheej)  feces  was  that  a  l)lug  of  plant  mateiial  foi'med  at  the  toj) 
of  the  centrifuge  tube  and  held  in  its  mesh  almost  all  the  fecal  mate- 
rial. The  feces  of  herbivora  are  composed  largely  of  plant  matter 
and  hence  largely  of  cellulose.  This  is  removable  to  a  great  extent 
by  sieving  but  is  not  at  all  soluble  in  Telemann's  reagents.  This  is 
also  true  of  the  feces  of  such  birds  as  chickens  and  pigeons,  as  tests 
with  such  feces  indicate. 

On  the  other  hand,  the  feces  of  man  and  of  the  carnivora  have  le-^s 
plant  matter  of  the  sort  and  more  matter  that  is  soluble  in  ether  and 
hydrochloric  acid.  Nevertheless,  comparative  tests  indicate  that  the 
writer's  method  is  practically  as  good  as  Telemann's  for  these  feces 
also.  In  tests  with  human  feces  where  the  writer's  method  gave  a 
theoretical  concentration  of  4:1,  Telemann's  method  gave  the  same 
result.  In  one  case  the  sediment  obtained  by  the  writer's  method  was 
treated  by  Telemann's  method,  and  after  sliaking  uj)  and  stirring 
was  again  centrifuged.  The  resultant  sediment  showed  no  reduction 
in  volume  as  a  result  of  the  action  of  the  chemicals.  In  anothei-  ca>e 
where  the  writer's  method  gave  a  sediment  of  O.8.")  cubic  centimetei-.  an 
application  of  Telemann's  method  to  this  sediment  reduced  it  to 
O.C)."*  cubic  centimeter.  Even  more  surprising  result-  were  obtained 
with  dog  feces,  used  as  rejiresentative  of  feces  of  the  carni\'ora.  In 
test  cases  sieving  in  water  gave  a  concentration  of  .") :  1.  In  one  case 
the  a])plication  of  Telemann's  method  to  the  sediment  so  oljtained 
reduced  it  to  half  of  its  volume,  thereby  doubling  the  concentration. 
Although  the  concentration  was  doubled,  the  fact  that  the  writer's 
method  had  reduced  the  original  1  cubic  centimeter  to  O.-i  cul)ic  centi- 
meter, thereby  eliminating  O.S  cubic  centimeter,  while  the  cheinicals 
had  onlv  reduced  it  bv  0.1  cubic  centimeter  more,  indicate-  that  the 


30  METHODS   OF   EXAMINING   FECES   FOR   PARASITISM. 

additional  solution  of  material  accomplished  by  the  use  of  these 
chemicals  was  but  slightly  more  than  the  solution  accomplished  by 
water  alone.  In  two  other  cases  where  dog  feces  had  been  allowed 
to  stand  a  long  time  in  water  and  had  been  shaken  up  for  four  or  five 
minutes  before  being  sieved  and  centrifuged,  the  resultant  sediment 
of  1.4  cubic  centimeters  was  actually  increased  after  the  application 
of  Telemann's  reagents  followed  by  centrifuging  to  a  flocculent  sedi- 
ment of  1.9  cubic  centimeters. 

The  results  of  comparative  tests  of  Telemann's  and  the  writer's 
method  were  quite  unexpected,  as  the  writer  had  used  Telemann's 
method  for  a  year  and  only  abandoned  it  when  its  inapplicability  in 
the  case  of  sheep  feces  became  to.o  evident  to  overlook.  As  has  been 
stated  previously  in  this  paper,  the  success  of  Telemann's  method  ap- 
pears to  be  due  more  to  the  efficiency  of  a  slight  solvent  action  in 
mechanically  breaking  up  feces  and  putting  them  in  condition  to 
sieve,  than  to  an  extensive  solvent  action.  Much  that  might  be  dis- 
solved in  the  chemicals  is  soluble  in  water,  much  that  might  be  dis- 
solved is  perhaps  taken  out  by  the  screen  where  Avater  alone  is  used, 
and  the  fact  that  the  slight  solvent  action  breaks  up  the  feces  and 
makes  them  easy  to  sieve  seems  to  be  the  feature  to  which  the  results 
obtained  by  this  method  are  to  be  attributed. 

The  feces  of  herbivora  have  little  material  that  is  soluble  in  Tele- 
mann's reagents,  and  the  breaking  up  secured  by  them  is  inferior  to 
that  secured  by  the  use  of  the  writer's  method.  Human  feces  break 
up  rapidly  in  Telemann's  reagents,  but  the  method  advocated  by  the 
writer  takes  about  the  same  time  and  gives  as  good  results.  Of  Tele- 
mann'^s  method.  Dock  and  Bass  (1910)  say: 

We  have  found  the  method  admirable  with  some  stools,  but  in  others  not 
enough  solution  occurs  to  permit  a  concentrated  layer  of  eggs  to  be  thrown 
down. 

Telemann's  method  has  one  disadvantage  which  neither  Telemann 
(1908),  Pfister  (1909),  nor  Quadflieg  (1909)  mentions  in  connection 
with  it — it  injures  the  microscope.  The  fumes  of  pure  hydrochloric 
acid  attack  the  lens  mountings  and  also  the  stage  and  the  lenses 
themselves.  The  use  of  vaseline  to  seal  the  edges  of  the  cover  glass 
affords  an  uncertain  protection,  and  the  use  of  high  powers  under 
such  circumstances  is  decidedly  unsafe.  In  the  writer's  opinion  the 
injury  to  the  microscope  would  of  itself  be  sufficient  reason  for  dis- 
carding this  method. 

CULTURE    METHODS. 

Quadflieg  (1909)  has  recommended  using  culture  methods  as  an 
aid  in  detecting  infection.  Tests  of  this  method  did  not  indicate  that 
it  added  to  the  certainty  of  detecting  infection,  though  live  embryo 
nematodes  could  be  detected  by  the  use  of  a  dissecting  microscope 


OBJECTIONS  TO   CERTAIN    METHODS.  31 

with  low-power  lenses.  As  the  deteriniiialion  of  iieiunlodc  ^-eiu'ra 
and  species  is  in  general  more  difficult  from  the  embryos  than  from 
the  eggs,  the  usefulness  of  the  culture  method  is  limited. 

Emerson  (1910)  uses  the  following  method  in  searching  for 
Stronyyloides  intestinalis : 

The  stool  should  be  placed  in  a  dish,  a  small  depression  made  on  its  surface, 
this  fllieil  with  water,  and  the  stool  left  in  a  thermostat  overnight.  If  embryos 
are  present,  they  are  easily  found     *     *     *     actively  swimming  in  this  water. 

Culture  methods  have  been  used  by  Ransom  (190Gi)  in  studying 
Hcemonchus  contortiis  in  sheep  feces.  Dr.  Garrison  tells  me  that  he 
has  seen  the  culture  method  used  successfully  in  Army  work  in  exam- 
ining a  company  for  hookworm  infection. 

It  is  evident  that  such  methods  are  suitable  only  for  special  pur- 
poses and  not  for  general  work. 

OBJECTIONS   TO   CERTAIN   METHODS. 

The  reason  for  rejecting  some  of  the  methods  outlined  in  the 
hypothetical  ideal  treatment  previously  mentioned  is,  as  was  stated, 
that  in  actual  practice  objections  of  two  general  kinds  may  be  urged 
against  them.  These  objections  are:  (1)  The  injury  to  parasites  or 
eggs;  (2)  the  unsuitability  of  the  methods  for  certain  kinds  of  feces 
or  the  fact  that  the  concentration  resulting  from  the  application  of 
the  method  may  be  too  insignificant  to  warrant  the  time  and  effort  ex- 
pended. It  will  be  fairly  evident  from  the  discussion  of  methods 
already  given  just  how  those  methods  fail  in  these  two  respects,  but 
a  brief  sunnnary  will  indicate  some  general  truths. 

INJURY  TO   1*.\RASITES. 

In  regard  to  the  first  point,  it  is  evident  that  if  parasite  eggs  or 
embryos  are  to  be  used  in  feeding  experiments  to  produce  infection, 
or  are  desired  alive  for  study,  or  if  the  movement  of  embryos  is  de- 
pended upon  to  indicate  viability  or  to  aid  the  eye  in  detecting  or 
locating  the  parasites,  certain  methods,  such  as  boiling  (Cobb,  1904) 
or  the  use  of  chemicals  which  will  kill  by  poisoning  or  by  rapid 
osmosis  (Telemann,  1908 ;  Bass.  1909) ,  can  not  be  used.  Bass's  ( 190!» ) 
method  has  the  further  objection  that  it  not  only  kills,  but  distorts. 
Letulle  (1905)  urges  that  certain  eggs,  such  as  those  of  Bilharzhi 
and  Vncinuria,  are  mechanically  injured  by  the  use  of  the  centrifuge. 
I  have  not  found  this  true  of  the  eggs  of  the  human,  .sheep,  or  dog 
hookworm,  or  any  other  parasites  encountered,  but  there  might  be 
cases  where  the  objection  would  hold  good.  The  writer's  use  of  shot 
would  work  occasional  injury  to  parasite  material  and  it  should  l>e 
avoided  when  possible.  In  almost  all  cases,  the  feces  can  be  bi'oken 
up  in  a  little  longer  time  by  shaking  in  water  alone,  as  experiments 
show. 


32  METHODS   OF   EXAMINING   FECES   FOR   PARASITISM. 

LIMITED    APPLICATION    OR     INCOMMf:NSrRATE    RESULTS. 

In  regard  to  the  second  point,  experience  shows  that  in  using 
specific  gravity  as  an  aid  in  concentration  it  is  safe  to  assume  that  tlie 
specific  gravity  of  a  parasite  is  greater  than  Avater,  though  all  eggs 
Avill  not  go  to  the  bottom,  as  some  are  held  up  by  lighter  material, 
bubbles,  etc.,  but  it  is  not  safe  to  rely  on  a  parasite  floating  on  a  solu- 
tion of  specific  gravit}'  greater  than  itself  for  an}'  length  of  time, 
owing  to  the  action  of  endosmosis.  An  additional  objection  to  this 
method  advocated  by  Bass  (1909)  is  that  experience  shows  that  all 
the  steps  are  not  warranted  by  the  results.  In  most  cases  the  simpler 
methods  used  by  the  writer  give  as  good  or  better  results.  The  use  of 
the  1.050  solution  appears  to  be  a  defect  of  the  method.  The  concen- 
tration attained  by  it  seems  trifling  in  an}-  case  and  at  times  it 
aj^parently  results  in  actual  loss  of  parasite  material.  Finally,  under 
the  same  class  of  objections,  experience  shows  that  the  concentra- 
tion obtained  by  the  use  of  chemicals  instead  of  water  (Telemann, 
1908)  is  no  greater  than  that  obtained  by  the  use  of  water  alone,  and 
hence  there  appears  to  be  no  reason  for  using  the  more  expensive 
chemicals,  especially  in  view  of  the  injury  to  the  microscope  which 
one  of  them  occasions.  It  might  also  be  urged  that  the  odor  of  these 
chemicals  is  such  that  one  would  prefer  to  avoid  them,  though  it  is 
also  true  that  they  deodorize  the  feces  after  a  fashion,  and  that  while 
they  kill  the  parasites  they  also  disinfect  the  feces  in  cases  where  the 
killing  is  immaterial, 

DISINFECTION  OF   FECES. 

Disinfection  and  deodorization  of  feces  can  be  more  easily  accom- 
plished, if  desired,  by  the  use  of  formol  solution  instead  of  water,  as 
suggested  b}^  Letulle  (1905),  Jones  (1907),  and  Garrison  (1910).  In 
a  discussion  of  this  point  before  the  Tlelminthological  Society  of 
"Washington,  December  1,  1910,  Dr.  Stiles  advocated  the  use  of  coal- 
tar  disinfectants  on  the  ground  that  protracted  work  with  formol 
material  would  cause  headaches.  The  writer  has  never  experienced 
any  inconvenience  from  the  use  of  formol.  Dr.  Stiles  also  pointed  out 
the  great  desirability  of  using  some  disinfectant,  for  the  reason  that 
the  greater  number  of  fecal  examinations  now  being  made  in  this 
country  are  for  evidence  of  hookworm  infection,  and  the  localities 
with  the  greatest  amount  of  hookworm  infection  have  a  high  typhoid 
index.  In  using  a  disinfectant,  the  writer's  method  of  comminution 
has  the  advantage  of  bringing  the  disinfectant  into  intimate  contact 
with  all  parts  of  the  fecal  mass.  The  thorough  breaking  up  of  the 
feces  and  the  use  of  large  amounts  of  water  reduces  tha  odor  to  a 
IDoint  where  it  is  almost  imperceptible  in  most  cases. 


SUMMARY.  33 


SUMMARY. 


After  tostinir  the  various  methods  as  above  indicated,  the  writer 
finds  tliat  the  best  results  in  routine  examinations  of  feres  of  all 
kinds  are  obtained  from  the  simple  method  already  ^iven.  Briefly, 
the  method  consists  in  breaking  up  the  feces  very  thoroughly  by  shak- 
ing in  Avater,  adding  a  quantity  of  small  shot  if  neeessaiy  or  desir- 
able; sieving  through  a  set  of  brass  sieves  and  then  through  a  silk 
bolting-cloth  sieve  or  a  sieve  made  with  a  jewehu-'s  line-meshed  i)rass 
screen,  examining  the  material  left  on  the  sieve  for  parasites:  sedi- 
menting  (and  washing)  ;  centrifuging  (and  washing) — one  tube 
being  filled  with  calcium  chlorid  solution  of  1.250  specific  grav- 
ity, centrifuged,  and  if  desired  the  top  cubic  centimeter  removed  with 
a  pipette,  shaken  up  in  a  tube  with  14  cubic  centimeters  of  watei-  and 
centrifuged — and  then  making  a  microscopic  examination  of  a  drop 
of  sediment  from  the  bottom  of  the  tube  centrifuged  with  water,  and 
one  from  the  to|)  when  the  calcium  chlorid  solution  alone  was  used  oi- 
from  the  bottom  in  case  water  was  added  to  the  to|)  cubic  centimeter. 
The  material  is  washed  at  either  or  both  of  the  points  indicated. 

ADAPTABILrrV    OF    METHOD. 

The  writer  does  not  claim  that  the  method  advocated  here  is  the 
best  possible  method.  It  is.  however,  the  method  which  his  experi- 
ence shows  to  be  the  best  for  routine  examination  of  various  kinds  of 
feces  after  comparative  tests  with  other  methods.  It  serves  very  well 
for  the  feces  of  man,  and  of  the  carnivora.  herbivora.  and  birds,  so 
far  as  fecal  examinations  for  representatives  of  the  last  three  groups 
indicate.  It  is  not  onh'  of  service  in  examining  feces  for  worm  para- 
sites, but  also  for  coccidia.  It  has  not  been  tested  for  other  protozoa. 
Presumably  the  writer's  conuninution  method  would  damage  flagel- 
lates, ciliates,  or  amel)a>.  It  is  often  useful  in  detecting  parasitic  in- 
fection of  st(miach  and  intestinal  contents.  It  has  (he  advantage  of 
speed  and  certainty  over  the  smear  method  or  sedimentation  methods. 
It  takes  longer  to  make  the  microscoj^ic  prei)aration  than  in  tiu^  smear 
method,  l)ut  the  resulting  concentration  justifies  it.  Xor  is  it  a  long  or 
difficult  i)rocess.  The  time  recpiired  for  each  stej)  is  slight — a  minute 
to  shake  up  the  feces,  another  to  sieve  them,  and  anothei"  to  centrifuge 
them,  leaving  out  the  sedimentation  after  sieving,  which  needs  no  at- 
tention, and  the  time  spent  in  examining  material  on  the  screens, 
which  examination  is  incidental  to  the  techni(iue.  not  part  of  it.  As 
everyone  who  has  used  laboratory  methods  knows,  the  total  time 
necessary  to  perform  three  one-minute  (►perations  is  not  just  three 
minutes  or.  as  a  rule,  even  six  minutes,  as  [)reparation.  intermediate 
steps,  cleaning  up.  etc..  add  considerably  to  the  time  noces>ai'y.  At 
the  same  time,  the  method  outlined  here  is  reasonably  ra|)id  and 
takes  less  time  than   is  required   to  examine  the  additional   slides 


34  METHODS   OF   EXAMINING   FECES   FOR   PARASITISM. 

which  the  use  of  the  smear  method  would  demand.  I  find  that 
the  careful  examination  of  a  slide  not  uncommonly  takes  9  or  10 
minutes.  Garrison,  Ransom,  and  Stevenson  (1903a)  state  that  the 
examination  of  100  preparations  is  an  average  day's  work  for  one 
person.  This  is  a  little  over  four  minutes  for  each  preparation. 
Stiles  (1909)  states  that  a  thorough  examination  of  10  slides  will 
take  40  to  CO  minutes,  or  4  to  G  minutes  for  each  preparation.  Dock 
and  Bass  (1910)  claim  to  look  over  an  ordinary  slide  thoroughly 
in  2  to  4  minutes.  If  one  slide  prepared  by  the  writers  method 
be  considered  as  equivalent  to  4  smear  preparations,  and  experi- 
ence shows  that  it  is  much  better  than  this,  then  it  will  save  time 
to  spend  8  minutes  in  obtaining  a  sediment  and  8  more  in  examin- 
ing 2  preparations  made  from  this  sediment,  rather  than  spend  82 
minutes  examining  8  smear  preparations.  "WHiere  positive  infor- 
mation as  to  i^arasitic  infection  is  desired,  the  best  methods  and 
the  time  necessary  for  these  methods  are  abundantly  warranted. 
Dock  and  Bass  (1910)  state  that  Gage  and  Bass,  in  the  examination 
of  the  feces  of  315  students,  found  only  47  per  cent  of  the  cases  of 
intestinal  parasitism  by  the  smear  method,  the  remaining  53  per  cent 
being  found  by  the  use  of  centrifuge  methods.  They  note  cases  where 
the  examination  of  25  smear  preparations  failed  to  show  infection, 
although  the  existence  of  infection  in  these  cases  was  demonstrated 
by  preparations  made  after  sedimenting  and  centrifuging. 

Only  in  those  cases  where  the  necessary  apparatus  for  better  meth- 
ods is  not  available,  or  where  evidence  of  heavy  infection  is  sought 
for  with  a  view  to  immediate  medical  treatment,  would  the  writer 
consider  the  routine  use  of  the  crude  smear  method  warranted.  In 
exceptional  cases  its  use  might  be  warranted  in  examining  one  or 
two  slides  for  specific  infection  where  there  is  likelihood  of  the 
infection  being  heavy  enough  to  be  promptly  discovered  by  this 
method. 

ECONOMY    OF    METHOD. 

If  the  time  of  a  physician,  veterinarian,  or  scientist  is  of  any  value, 

then  the  smear  method  is  not  even  more  economical  than  the  writer's 

method.    The  centrifuge  is  a  thing  which  the  workers  just  mentioned 

should  have  for  purposes  other  than  fecal  examinations,  and  the  only 

additional  pieces  of  apparatus  required — the  screens — are  inexpensive 

and  durable. 

CONCLUSION. 

The  method  of  examining  feces  for  evidences  of  parasitism  which 
consists  in  putting  the  feces  through  a  process  of  thorough  shaking, 
sieving,  sedimenting,  and  centrifuging  appears,  from  a  theoretical 
standpoint  and  in  actual  experience,  to  give  the  best  results  in  the 
shortest  time  and  with  a  mininuun  financial  expenditure  when  the 
value  of  time  saved  is  considered.  It  is  therefore  advocated  as  a 
practical  routine  method  of  examining  feces  of  all  sorts. 


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'  This  biblioKrapliy   is  prepared  in  tlie  st.vle  used   in  Itnlletin  .".n  of  tliis  bureau.  Index- 
Catalogue  of  Medical  and  Veterinary  Zoology. 


36  METHODS   OF   EXAMINING  FECES   FOR   PARASITISM. 

QUADFLIEG,  L.      [Dr.] 

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Ransom,  B.  H.     [B.  Sc,  A.  M.,  Ph.  D.] 

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